The present invention relates to the culturing of mammalian cell types and more particularly to the use of novel structures for culturing mammalian cells in submerged culture.
It is a common procedure to use various single cells microorganisms, such as yeast and bacteria, in a liquid-nutrient medium for the production of various fermentation products. More recently there has been an an increase in the exploitation of hybridoma, animal, plant, insect and genetically engineered mammalian cells to obtain pharmacologically and diagnostically useful products. The use of mammalian cells to produce pharmaceutical and diagnostic valuable products has resulted in efforts to culture mammalian cells on a large scale.
As mammalian cells are more fragile than microorganisms, they must be grown under conditions which avoid the shear forces associated with turbulence encountered in the well developed technology of industrial microbiology for culturing microorganisms. To avoid excessive shear forces during cell growth, mammalian cells, and particularly anchorage dependent cells (ADC), have traditionally been grown on a large scale on the inside walls of rotating bottles. In such instances, growth of relatively thin layers of cells on the inside surface of these bottles represents only a small fraction of the bottle volume. To increase the cell density within a reactor vessel, hollow fiber reactors, consisting of a bundle of hollow fibers surrounded by a shell, have been used for growing various mammalian cells. See, for example, U.S. Pat. Nos. 3,883,393; 3,997,396; and 4,804,628.
A major limitation of hollow fiber culture devices is that as the cells grow and the cell density increases, it becomes more difficult for the nutrients. oxygen, and other chemical stimuli in the growth medium to diffuse through the walls of the hollow fiber membranes and through the layers of cells that have developed to reach remote cells (cells which are furthest away from the reactor membranes). It also becomes equally difficult for waste material produced by the remote cells to diffuse back into the lumen of the hollow fiber membranes. Other limitations include fiber leaks, poor accessibility to the extracapillary spaces, an undesirable pressure drop across the device as it is scaled up, and the fibers sticking together reducing the surface area to which the cells may attach.
To overcome the limitations inherent in the previous systems, cells have been attached to various carrier systems. Solid microcarriers fabricated from glass, silica, polystyrene, cross-linked dextran or polyhydroxyethylmethacrylate have been used for the cultivation of cells. See, for example, M. Kiremitci et al., Enzyme Microb. Technol. 11, 205-224 (1989): S. Reuveny, Adv. Biotechnol. Proc. 2, 1-32 (1980). Polystryene resin beads derivatized with amino acids, peptides, or hydroxy carboxylic acids and their use in culturing cells is described in U.S Pat. No. 4,266,032. British Patent 2,178,447 describes fiber networks or open pore foams having open areas or pores in which cells can be grown. However, as mammalian cells attach and grow on the outer surfaces of a microcarrier system, it is still necessary to avoid excessive shear forces encountered at mixing rates necessary to promote adequate nutrient and oxygen transfer in large scale reactors.
Another disadvantage of using various carrier systems for mammalian cells, and particularly ADC, is the inoculum size required. This is of particular importance as cells will attach to the outer surfaces of the carrier as well as within the pores and internal structures when the cells are incubated with the carrier at low mixing speeds. The cells attached to the outside surface will often be destroyed by high shear forces when agitation is increased to promote adequate nutrient and oxygen transfer.
It would therefore be desirable to provide simple protective structures in which mammalian cells can be grown. Structures having uniform dimensions which promote consistent cell growth but which can be fabricated at low cost are particularly desirable. In addition, for large scale production of products by mammalian cells it would be desirable to reduce the number of cells and manipulative steps necessary for inoculation.